Low temperature albumin fractionation using sodium caprylate as a partitioning agent

ABSTRACT

Highly stable plasma-derived therapeutic albumin solutions, having a turbidity level of 5 NTU or less can be made by adding sodium caprylate to Cohn fraction II+III or IV-1 effluent at relatively low temperatures. The sodium caprylate acts as a partitioning agent to separate albumin from unwanted proteins. In preferred embodiments, the albumin source solution temperature is elevated, increased in pH and reacted for approximately six hours under conditions sufficient to disrupt the initial solution colloid, and partition albumin-containing supernatant from a colloidal disperse phase, which retains unwanted globulins and manufacturing debris. Since it tends to be a scavenger molecule, albumin is selectively stabilized by diafiltration against a buffer containing sodium caprylate, thereby assuring a high albumin monomer content and low turbidity level. The amount of sodium caprylate required for selective stabilization is determined by the amount of available binding sites on the albumin molecule.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This disclosure generally involves manufacturing plasma-derivedtherapeutic protein solutions, and more specifically, manufacturingselectively stabilized animal or human serum albumin (HSA), Alpha-1Protease Inhibitor (Alpha-1 PI), and Antithrombin III (AT III) fromserum or plasma.

2. Prior Art Description

The Cohn fractionation method, which utilizes ethanol, temperature, pH,protein concentration, ionic strength, and time to insolubilize unwantedproteins during albumin manufacture, was originally published in 1946,and remains a primary method in the United States for processing plasma.Cohn et al., J. Am. Chem. Soc. 68, 459 (1946). T. Gerelough's subsequentuse of 95% ethanol in the Cohn fractionation process greatly diminishedprocess volumes required, and thereby reduced correspondingmanufacturing costs. Gerelough's method is also a recognized standard inthe United States for plasma fractionation. U.S. Pat. Nos. 2,710,294;2,710,293 (1955). In Europe, H. Nitschamann and P. Kistler describe ashorter method for processing albumin; however, the resulting productfailed to satisfy regulatory guidelines imposed by United Statesagencies during that time period. Vox Sang., 5, 272 (1960).

Plasma fractionation methods in the United States have employed Cohntechniques since Cohn et al's. 1946 publication. Consequently, neitherinterest nor necessity encouraged manufacturers to identify alternativealbumin fractionation techniques for more than twenty years. M.Steinbuch, Vox Sang., 23, 92 (1972). Furthermore, since conventionalfractionation techniques produced albumin that could be successfullypasteurized (by heating for 10 hours at 60 degrees centigrade) toinactivate viruses, there was little motivation and much caution amongalbumin manufacturers in seeking alternative and improved fractionationmethods.

Then in 1972 M. Steinbuch explored the ability of several reagents,other than ethanol, to separate plasma proteins via precipitation. UsingCohn Fraction III as starting material, Steinbuch studied theprecipitation capacity of caprylic acid, which had previously been usedto stabilize albumin (M. Steinbuch, Vox Sang. 23:92-106, 1972, Yu L.Hao, U.S. Pat. No. 4,222,934, 1980), and subsequently to inactivatelipid-enveloped viruses. Seng et al., U.S. Pat. No. 4,939,176, 1990. Asa result of these studies, scientists later developed several techniquesfor purifying IgG, IgA, alpha-1 acid glycoprotein and prealbumin,concurrently finding that the precipitation reaction was highlytemperature, and pH dependent.

During human immunoglobulin preparation caprylic acid is generallyrecognized as an effective precipitating agent for most plasma proteinsat pH 4.8, so long as parameters such as temperature and ionic strengthare optimized. Steinbuch et al., Preparative Biochemistry, 3(4), 363-373(1973). Accordingly, Steinbuch et al. have described a method forisolating IgG from mammalian sera using caprylic acid, finding thatextensive non-immunoglobulin precipitation is best obtained at slightlyacidic ph, but not below pH 4.5. Steinbuch et al., Arch. Biochem.Biophys., 134, 279-294 (1969).

Habeeb et al. used caprylic acid precipitation to obtain plasma-derivedIgG that was free of aggregates, plasmin and plasminogen; low inanticompliment activity; and stable during storage. PreparativeBiochemistry, 14(1), 1-17 (1984). Also, IgA has been prepared as aroutine fractionation by-product from Cohn fraction III, based on IgAsolubility with caprylic acid present at pH 4.8. Pejaudier et al., VoxSang. 23, 165-175 (1972). Fraction III additionally provides startingmaterial for obtaining IgM-enriched plasma fractions.

Sodium caprylate has also been used to purify albumin. According tothese methods, sodium caprylate is added to process plasma, and protectsalbumin when the process stream is exposed to high temperatures. Extremetemperatures not only denature process stream globulins, but oftengenerate contaminant neo-antigens. Schneider et al., U.S. Pat. No.4,156,681 (1979); Institute Merieux, U.S. Pat. No. 3,992,367.

Cohn fraction III was also treated with caprylic acid to precipitateceruloplasmin, an alpha globulin that facilitates plasma coppertransport. Employing this technique to obtain ceruloplasmin avoideddenaturation steps involving ethanol or acetone, but has been obtainedin this way only from horse, mule, rabbit, goat, sheep, and baboonplasma. M. Steinbuch, Vox Sang., 23, 92-106 (1972).

Currently, technical and patent literature contain numerous albuminmanufacturing methods that incorporate sodium caprylate as a stabilizingagent, and purification techniques involving caprylic acid precipitationto obtain immunoglobulins from plasma-derived Cohn fraction III.However, we are unaware of disclosures employing sodium caprylate as apartitioning agent during albumin manufacture to separate albumin fromunwanted globulins and manufacturing debris.

Unexpectedly, we have also discovered significant advantages inmanufacturing albumin with sodium caprylate to partition albumin fromunwanted material, instead of using an ethanol precipitant. First,sodium caprylate partitioning shortens conventional albuminmanufacturing methods, which reduces product handling andcorrespondingly improves albumin recovery by 25% or more. Resultingalbumin yields are essentially aluminum-free, and exhibit 97% or greatermonomer levels. Second, sodium caprylate partitioning significantlyreduces the time needed to complete albumin manufacture, thus reducingequipment-related manufacturing costs by at least 65%. Third, sodiumcaprylate partitioning improves Alpha-1 PI and AT-III yields from Cohnfraction II+III, and is generally more energy efficient thanconventional methods of manufacturing plasma products. Finally, andperhaps most important, sodium caprylate partitioning significantlyreduces ethanol use, and completely eliminates the use of acetone duringalbumin manufacture, which largely avoids polluting our environment withnoxious and environmentally harmful solvent residues. Our findings arediscussed and illustrated in detail below.

SUMMARY OF THE INVENTION

The invention provides a method for manufacturing essentially monomeric,aluminum-free albumin using sodium caprylate as a partitioning agent.

The use of sodium caprylate intercepts conventional plasma fractionationat either the Cohn fraction II+III or fraction IV-1 effluent step.According to the invention, sodium caprylate is added to a colloidaleffluent material including the desired albumin, and the undesirednon-albumin proteins and contaminants.

After temperature and pH are elevated, the mixture then incubates forapproximately 6 hours, allowing sodium caprylate to act as apartitioning agent by breaking the colloid solution into a supernatantcontaining albumin, and a disperse phase containing unwanted non-albuminproteins (e.g., globulins), and manufacturing debris. The sodiumcaprylate-treated suspension is then centrifuged, and deae sephadex isadded to assist in filtration. The suspension is next filtered,ultrafiltered to 12% protein, diafiltered with 0.02M sodium caprylate,harvested, and bulked for sterile filtration. When the sterile bulkpasses sterility testing, the albumin is filled in final containers.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2: Flow Charts

The figures are flow charts distinguishing the present invention fromconventional albumin manufacture according to Cohn fractionationmethodology. The flow chart emphasizes only those Cohn fractionationsteps necessary to differentiate the disclosed, abbreviated albuminmanufacturing method from protracted Cohn fractionation techniques.Processing hours for the prior art method are based on 20 Sharplescentrifuges, and most other times are based on 1000 liters of in-processplasma.

DEFINITION OF TERMS

1. Partitioning Agent, as used herein, means a substance that, whenadded to plasma protein mixtures during albumin manufacture, generates asuspension colloid consisting of two separate phases: a supernatant thatcontains albumin; and an opalescent disperse phase containingagglomerated protein particles, including alpha and beta globulins.

2. Precipitating Agent, as used herein, means a substance that, whenadded to plasma protein mixtures during albumin manufacture, is capableof reversibly insolubilizing solution material when the solution pH iswithin a specific range.

3. Non-Albumin Proteins, as used herein, means all non-albumin proteins,primarily alpha, beta and gamma globulins, and acid glycoproteins.

4. Manufacturing Debris, as used herein, means non-protein contaminants,and primarily includes multivalent metal ions, such as aluminum, andmanufacturing solvents, such as ethanol.

5. Albumin-Containing Solution, as used herein, means either Cohnfraction II+III effluent, or Cohn fraction IV-1 effluent.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

MATERIALS AND METHODS

Materials:

1. Starting material for the disclosed invention, Cohn fraction IV-1effluent, was produced according to the conventional Cohn plasmafractionation method, and derived from source plasma, fractionated byMiles Inc. in Clayton, N.C.

2. Source plasma was produced from fully screened plasma according toMiles' current screening procedure, which was performed within Miles'licensed Clayton, N.C. facility.

3. Only terminally-heated samples were evaluated to support thisprocess. Those skilled in the art realize that protein shifts during theterminal treatment of pasteurization cannot be anticipated with today'stesting methodology.

4. Testing required by the CBER and foreign government regulationsprovided final container testing criteria.

Methods:

1. Purity Determinations

A. Cellulose Acetate Electrophoresis (CAE) was used to the presence ofproteins other than albumin.

B. High Performance Liquid Chromatography (HPLC) was used to determinethe presence of aggregates, and other protein molecular weights.

C. Albumin filtrate turbidity was measured in National Turbidity Unitsusing a Hach nephelometer.

EXAMPLE I

The improved albumin manufacturing method begins with four standard Cohnfractionation steps. First, human plasma is pooled, thawed, andcentrifuged. The formed cryoprecipitate is harvested and processed toproduce factor VIII concentrate, while the effluent's temperature isreduced to about minus 2 degrees centigrade while adding 95% ethanolcontaining pH 4.0 acetic acid buffer (Buffer A).

When alcohol addition is complete, the resulting suspension, Cohnfraction I, is approximately 8% ethanol by volume, and at pH 7.3 whendiluted 1:5 with saline or distilled water. A majority of plasmafibrinogen is precipitated within a two hour reaction period.

Fraction I 8% is centrifuged to remove the formed solids. The effluentFraction I is then brought to Fraction II+III 20% by the slow additionof ethanol containing buffer A, and the material's temperature islowered to about minus 5 degrees centigrade. The final plasma pH isabout 6.8. After an approximate 2 hour reaction time, Fraction II+III20% is centrifuged, and crude II+III paste containing the gamma globulinfraction is isolated. This paste is later processed to manufacture IGIVand ISG.

The resulting Cohn Fraction II+III effluent is next treated with coldacetic acid buffer, reaching a suspension pH of about 5.2 when diluted1:10 with distilled water. Effluent is incubated for about a 6 hourreaction period, which incorporates precipitation and denaturization.Then alpha globulins and other insoluble proteins are harvested, and theresulting precipitate is used to manufacture Alpha-I PI and AT III.

According to conventional fractionation methods, Fraction IV-1 effluentis further processed to Cohn Fraction IV-4, primarily to remove heatunstable alpha and beta globulins, and then undergoes four subsequentethanol precipitations before either acetone drying, lyophilization,thin film evaporation, or ultra and diafiltration.

According to the present invention, sodium caprylate is next added toCohn fraction IV-1 effluent, which insolubilizes alpha and betaglobulins by wetting, or partitioning albumin from these unwantedproteins. Sodium caprylate also functions as an antiviral agent, andadditionally permits mechanical separation of albumin.

Approximately 10 grams of sodium caprylate per liter is added tofraction IV-1 effluent, which is heated to about 25 to 35 degreescentigrade while simultaneously increasing solution pH to about 5.4 to5.8. The reaction is completed within a time period of 6 or more hours,during which the pH is maintained at about 5.3 to 5.6, but preferably at5.4.

Increasing the solution's temperature helps dissolve sodium caprylate tocomplete the reaction. Increasing the pH improves albumin recovery sincepH levels lower than about 5.4 approach albumin's isoelectric range,which is less than the preferred pH, and subsequently could result inalbumin loss. However, a pH level greater than about 5.8 may solubilizeheat unstable globulins, and thus permit their escape into the finalproduct. Total incubation time following sodium caprylate addition isapproximately 6 hours.

The sodium caprylate-treated solution is then cooled to about 18 degreescentigrade or colder to inhibit bacterial growth, and centrifuged.Afterwards, about 1 gram of Deae Sephadex is added to the effluent toaid filtration. The caprylate effluent is then clarified through 0.2micron depth and membrane filters, which produce the deae filtrate. (SeeFIG. 1). The resulting filtrate pH is then increased to neutrality (pH6.8 to 7.2) with sodium carbonate.

According to conventional methods, when acid filtrate pH is increased toneutrality, solution turbidity improves. Turbidity levels of 12 NTU andhigher commonly occur at pH 5.5, and decrease to about 8 NTU when pH iselevated above 5.5. This phenomena is caused by departing from thecontaminating globulin's isoelectric point.

However, according to the disclosed invention, Deae filtrate turbiditiesare about 3 NTU or less, and when pH is elevated to 6.8, there is nosignificant turbidity level change, even after 10 or more hours at 60degrees centigrade.

Thus, increasing the sodium caprylate-treated filtrate pH to neutralitydoes not affect turbidity levels. Rather, filtrates resulting fromsodium caprylate-treated IV-1 effluent maintain a turbidity level of 5NTU or lower, even at an elevated pH, since filtrates are essentiallyfree of contaminant globulins after incubation with sodium caprylate.

The Deae-Sephadex clarified filtrate is then ultrafiltered withRhomicon-type ultrafilters, and diafiltered against at least sevenvolume exchanges of sodium caprylate diafiltration buffer to removemetal contaminants, ethanol and salts. The diafiltration buffer isprepared according to the final container albumin concentration. Forinstance, if the desired final albumin concentration is 25%, then 0.02Msodium caprylate diafiltration buffer is used. If the final albuminconcentration is to be 5%, 0.004M sodium caprylate diafiltration bufferis needed. Using 30,000 molecular weight (MWCO) ultrafiltration mediaprovides excellent flux rates.

Albumin is then ultrafiltered using conventional techniques to achievethe desired final container albumin concentration. The targetconcentration must be sufficient to permit an equipment rinse down withdiafiltration buffer and thereafter yield 25%, 20%, 7% or 5% finalalbumin concentrations. Finally, the concentrate is sterile filtered,bulked for release testing, and filled into final containers.

Among the many conditions affecting this invention are temperature, pH,sodium caprylate concentration, and reaction time. An experiment wasconducted on Cohn fraction effluent IV-1, varying one parameter at atime to establish the ideal temperature of the caprylate reaction.Temperatures in the range of twenty degrees centigrade were tried andfound to produce a turbid final container. Also, a reaction time of sixhours was determined arbitrarily, since processing large albumin volumesrequires varying time needed to complete the reactions. The inventionwas dependent on the aforementioned tests, and the effects of heat onalbumin.

EXAMPLE II

Bench lots were composed of 100 to 200 liters of Cohn Fraction IV-1effluent. Several bench lots were processed to achieve desirableparameters. The final test of acceptance was based on turbidities afterthe albumin was heated 10 hours at 60 degrees centigrade.

A scaled up lot of albumin was processed from Cohn Fraction IV-1. Inthis lot, 1100 liters of effluent Cohn Fraction IV-1 was processed to25% albumin final containers using the method described in Example 1.

RESULTS

The process herein described yielded the following data with scale up:

Protein Concentration: 24.03% Protein

CAE: 100% Albumin

pH: 6.78

Heat Stability Duplicate

(50 Hours@57): Pass

Aluminum: 7.53 ppb

PKA: 1% Of Reference

Citrate: Less Than 5 ppm

Turbidity: 2.6 NTU

Sodium: 152 Meq/L

HPLC:

Monomer: 97.58%

Dimer: 2.42%

Viscosity: 7.98 CPS

Density: 1.0699

Pyrogen (3 Rabbits): 0.2 Total Of All

Caprylate: 0.086M Selectively Bound*

* A two volume CWFI diafiltration would reduce this level to about 0.07molar.

DISCUSSION

We have demonstrated that human serum albumin, a widely used therapeuticreagent, can be manufactured faster, more efficiently, and for lessmoney by using sodium caprylate to partition albumin from unwantedproteins and manufacturing debris. Additionally, sodium caprylatealbumin fractionation as described here avoids the use of solvents suchas acetone, thus avoiding polluting the environment with chemicalsolvents; reduces manufacturing and equipment costs; decreases albuminfinal container production time; and increases the yield of albumin, andother plasma-derived products such as Alpha-1 PI and AT III.

Furthermore, we discovered that albumin naturally selects and binds,through molecular attraction, the amount of sodium caprylate retained infinal albumin solutions. Following sodium caprylate addition, thealbumin solution's turbidity level remains below 5 NTU even aftersubsequent filtration, ultrafiltration, diafiltration, andpasteurization at 60 degrees for 10 hours. Consequently, sodiumcaprylate enhances product stability during the manufacturing process,as evidenced by low turbidity levels after adding sodium caprylate; andprotects albumin from thermal breakdown during terminal high heatpasteurization, which prevents increasing turbidity during long-termstorage.

Conventional albumin manufacturing methods commonly employacetyl-dl-tryptophan, a suspected carcinogen, or sodium chloride tostabilize albumin. However, neither the tryptophan nor sodium chlorideare capable of enhancing both short, and long-term albumin stability.Albumin does not bind to tryptophan, but tryptophan nonetheless protectsalbumin's structural integrity during exposure to high temperatures.Conversely, albumin binds strongly to sodium chloride, but sodiumchloride fails to protect albumin during exposure to high temperatures.Thus, in addition to partitioning albumin from unwanted material, sodiumcaprylate serves the dual function of improving and maintaining albuminstability both during, and after the manufacturing process.

In light of the examples and discussion above, several potentialmodifications and variations of the disclosed invention will occur tothose skilled in the art. Consequently, the examples provided merelyillustrate the invention, which should be limited only by the followingclaims.

We claim:
 1. In a method of preparing albumin from a solution of plasmaproteins that contains albumin, non-albumin proteins and contaminantmanufacturing debris including metal ion contaminants, ethanol andsalts, the improvements comprising the steps of(a) employing sodiumcaprylate in the solution of plasma proteins at a pH of about 5.25 to5.6 as a partitioning agent to separate albumin from the non-albuminproteins; (b) separating the albumin of step (a) from the non-albuminproteins; and (c) diafiltering the separated albumin of step (b) againstsodium caprylate diafiltration buffer to remove metal ion contaminants,ethanol and salts.
 2. The method, according to claim 1, wherein sodiumcaprylate is added to the solution of step (a) at a temperature rangingfrom about 20 to 30 degrees centigrade.
 3. The method, according toclaim 2, where in the sodium caprylate-treated solution of step (a) isincubated for a period ranging from about 2 to 8 hours.
 4. The method,according to claim 1, wherein sodium caprylate is added to the solutionof step (a) in an amount ranging from about 0.04M to 0.08M sodiumcaprylate.
 5. The method, according to claim 1, wherein said solution ofplasma proteins is brought to a pH level between about 5.4 and 5.6,elevated to a temperature between about 20 and 30 degrees centigrade,and mixed while sodium caprylate is added to achieve a concentration ofbetween about 0.04M to 0.08M, and incubated for a period of betweenabout 2 to 8 hours.
 6. The method of claim 5 wherein the solution ofstep (a) is brought to about pH 5.4, heated to about 30 degreescentigrade, treated with approximately 0.06M sodium caprylate andincubated for about 6 hours.
 7. The method, according to claim 1,wherein said solution of plasma proteins comprises either Cohn fractionII+III effluent or Cohn fraction IV-1 effluent.
 8. The method, accordingto claim 1, wherein said solution of plasma proteins comprises Cohnfraction IV-1 effluent.
 9. The method, according to claim 1, wherein thesolution of plasma proteins comprises Cohn fraction II+III effluent.